Human preprotachykinin B

ABSTRACT

The invention provides a human preprotachykinin B (PPT-B) and polynucleotides which identify and encode PPT-B. The invention also provides expression vectors, host cells, agonists, antibodies and antagonists. The invention also provides methods for treating disorders associated with expression of PPT-B.

This application is a divisional application of U.S. application Ser.No. 08/879,995, filed Jun. 19, 1997.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of ahuman preprotachykinin B and to the use of these sequences in thediagnosis, prevention, and treatment of neurological and neoplasticdisorders.

BACKGROUND OF THE INVENTION

Biologically active peptides function as hormones, paracrine regulators,or chemical neurotransmitters. These peptides are usually released byproteolysis of a larger, often inactive, precursor molecule.Neuropeptides perform many functions in the central nervous system (CNS)as neurotransmitters, neuromodulators, and neurotrophic factors(Stewart, J. M. and Hall, M. E. (1993) Agents Actions Suppl.42:211-226). Neuropeptides are also important regulators of amineneurotransmitter release and can be identified as playing importantroles in several pathological states (Stewart and Hall (1993) supra).

The tachykinins comprise a family of closely related peptides thatparticipate in the regulation of diverse biological processes. They arecharacterized by the amino acid residue sequence-Phe-X-Gly-Leu-Met-NH₂at the C-terminus in which X represents an aromatic (Phe, Tyr) orbranched aliphatic (Val, Ile) amino acid. The tachykinin peptidessubstance P (SP), neurokinin A (NKA), NKA(3-10), neuropeptide K, andneuropeptide γ are produced from a single preprotachykinin gene,preprotachykinin A (PPT-A). Differential splicing of PPT-A mRNA yieldsαPPT-A, βPPT-A, γPPT-A, and δPPT-A mRNA species. Postranslationalprocessing of the preproprotein gives rise to multiple active productsboth within a single cell and in different cells expressing the gene(c.f. Helke, C. J. (1990) FASEB J. 4:1606-1615; Nakanishi, S. (1987)Physiol. Rev. 67:1117-1142). N-terminally extended forms of SP and NKA,but not the mature peptides, are detected in measurable amounts in humancerebrospinal fluid (CSF) using high performance liquid chromatography(HPLC). It has been suggested that their levels in CSF can be used asmarkers of the activity in central SP and NKA neurons (Toresson, G. etal. (1993) Regul. Pept. 46:357-359). In mammals, SP and NKA causevasodilation in the circulatory system, are involved in inflammation andimmune processes, and play a role in the pathogenesis of allergicdiseases (Otsuka, M. and Yoshioka, K. (1993) Physiol. Rev. 73:229-308).

A second preprotachykinin gene, PPT-B, encodes proneurokinin B (Kotani.H. et al. (1986) Proc. Natl. Acad. Sci. USA 83:7074-7078). Proteolyticcleavage of rat proneurokinin B yields neurokinin B (NKB) and a30-residue peptide (Lang, S. and Sperk, G. (1995) Regul. Pept.57:183-192). PPT-B mRNA and peptide products are differentiallydistributed throughout the CNS (particularly in the hypothalamus) andperipheral tissue (Helke, C. J. (1990) supra). PPT-B mRNAs have beenisolated from bovine and rat brain, and the proteins share 75% aminoacid residue identity (Kotani, H. et al. (1986) supra; Bonner, T. I. etal. (1987) Brain Res. 388:243-249).

NKB has been shown to be involved in a broad range of biologicalfunctions. Foe example, acute treatment of rats with synthetic rat NKBprevented a decline in cortical choline acetyltransferase activityassociated with injection of N-methyl-D-aspartate into the nucleusbasalis magnocellularis. NKB also attenuated impaired behavioralperformance produced by entorhinalcortex lesions (Wenk, G. L. et al.(1997) Behav. Brain Res. 83:129-133). Levels of rat NKB mRNA andNKB-immunoreactivity in the granule cells of the rat hippocampus wereenhanced following limbic epileptogenesis. It has been suggested thatthese changes may have profound effects on synaptic transmission andcontribute to modulate hippocampal excitability (Schwarzer, C. et al.(1996) Brain Res. Brain Res. Rev. 22:27-50). Large amounts of NKB, butnot of SP, or NKA, were found in tumors of the peripheral nervous systemand it has been suggested that PPT-B gene expression predominates overthat of PPT-A and may be used as a tumor marker of nervous tissue(McGregor, G. P. et al. (1990) FEBS Lett. 277:83-87). NKB induced airwaymucus secretion in the rat, and an NKB agonist, [MePhe7]-NKB, causedcontraction of guinea pig lung parenchymal strips in vitro (Wagner, U.et al. (1995) Life Sci. 57:283-289; Killingsworth, C. R. and Shore, S.A. (1995) Regul. Pept. 57:149-161). NKB modulates cellular biochemistryby interacting with NK-3, a G-protein coupled receptor, inducing secondmessenger pathways activated by Ca²⁺, inositoltrisphosphate, anddiacylglycerol (Helke, C. J. et al. (1995) supra). The properties of the30 residue peptide (preprotachykinin B(50-79)) are unknown.

The discovery of a new human preprotachykinin B and the polynucleotidesencoding it satisfies a need in the art by providing new compositionswhich are useful in the diagnosis, prevention and treatment ofneurological and neoplastic disorders.

SUMMARY OF THE INVENTION

The invention features a substantially purified polypeptide, humanpreprotachykinin B (PPT-B), having the amino acid sequence shown in SEQID NO:1, or fragments thereof.

The invention further provides an isolated and substantially purifiedpolynucleotide sequence encoding the polypeptide comprising the aminoacid sequence of SEQ ID NO:1 or fragments thereof and a compositioncomprising said polynucleotide sequence. The invention also provides apolynucleotide sequence which hybridizes under stringent conditions tothe polynucleotide sequence encoding the amino acid sequence SEQ IDNO:1, or fragments of said polynucleotide sequence. The inventionfurther provides a polynucleotide sequence comprising the complement ofthe polynucleotide sequence encoding the amino acid sequence of SEQ IDNO:1, or fragments or variants of said polynucleotide sequence.

The invention also provides an isolated and purified sequence comprisingSEQ ID NO.2 or variants thereof. In addition, the invention provides apolynucleotide sequence which hybridizes under stringent conditions tothe polynucleotide sequence of SEQ ID NO:2.

In another aspect the invention provides a composition comprising anisolated and purified polynucleotide sequence comprising the complementof SEQ ID NO:2, or fragments or variants thereof. The invention alsoprovides a polynucleotide sequence comprising the complement of SEQ IDNO:2.

The present invention further provides an expression vector containingat least a fragment of any of the claimed polynucleotide sequences. Inyet another aspect, the expression vector containing the polynucleotidesequence is contained within a host cell.

The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a fragment thereof,the method comprising the steps of: a) culturing the host cellcontaining an expression vector containing at least a fragment of thepolynucleotide sequence encoding PPT-B under conditions suitable for theexpression of the polypeptide; and b) recovering the polypeptide fromthe host cell culture.

The invention also provides a pharmaceutical composition comprisingsubstantially purified PPT-B having the amino acid sequence of SEQ IDNO:1 in conjunction with a suitable pharmaceutical carrier.

The invention also provides a purified antagonist of a polypeptide ofSEQ ID NO:1. In one aspect the invention provides a purified antibodywhich binds to a polypeptide comprising at least a fragment of the aminoacid sequence of SEQ ID NO:1.

Still further, the invention provides a purified agonist which modulatesthe activity of the polypeptide of SEQ ID NO:1.

The invention also provides a method for treating or preventing aneurological disorder comprising administering to a subject in need ofsuch treatment an effective amount of a pharmaceutical compositioncontaining PPT-B.

The invention also provides a method for treating or preventing aneoplastic disorder comprising administering to a subject in need ofsuch treatment an effective amount of an antagonist to PPT-B.

The invention also provides a method for detecting a polynucleotidewhich encodes PPT-B in a biological sample comprising the steps of: a)hybridizing the complement of the polynucleotide sequence encoding PPT-Bto nucleic acid material of a biological sample, thereby forming ahybridization complex; and b) detecting the hybridization complex,wherein the presence of the complex correlates with the presence of apolynucleotide encoding PPT-B in the biological sample. In one aspect,prior to hybridization, the nucleic acid material of the biologicalsample is amplified by the polymerase chain reaction.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show the amino acid sequence (SEQ ID NO:1) and nucleicacid sequence (SEQ ID NO:2) of human PPT-B. The alignment was producedusing MACDNASIS PRO software (Hitachi Software Engineering Co. Ltd. SanBruno, Calif.).

FIG. 2 shows the amino acid sequence alignments among human PPT-B (SEQID NO:1), bovine PPT-B (GI 163590; SEQ ID NO:3), and rat PPT-B (GI205724; SEQ ID NO:4), produced using the multisequence alignment programof DNASTAR software (DNASTAR Inc, Madison Wis.).

FIGS. 3A and 3B show the hydrophobicity plots for human PPT-B, (SEQ IDNO:1) and bovine PPT-B (SEQ ID NO:3), respectively. The positive X axisreflects amino acid position, and the negative Y axis, reflectshydrophobicity (MacDNASIS PRO software).

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms "a", "an", and "the" include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to "ahost cell" includes a plurality of such host cells, reference to the"antibody" is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

Definitions

PPT-B, as used herein, refers to the amino acid sequences ofsubstantially purified PPT-B obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

The term "agonist", as used herein, refers to a molecule which, whenbound to PPT-B, increases or prolongs the duration of the effect ofPPT-B. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to and modulate the effect of PPT-B.

An "allele" or "allelic sequence", as used herein, is an alternativeform of the gene encoding PPT-B. Alleles may result from at least onemutation in the nucleic acid sequence and may result in altered mRNAs orpolypeptides whose structure or function may or may not be altered. Anygiven natural or recombinant gene may have none, one, or many allelicforms. Common mutational changes which give rise to alleles aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

"Altered" nucleic acid sequences encoding PPT-B as used herein includethose with deletions, insertions, or substitutions of differentnucleotides resulting in a polynucleotide that encodes the same or afunctionally equivalent PPT-B. Included within this definition arepolymorphisms which may or may not be readily detectable using aparticular oligonucleotide probe of the polynucleotide encoding PPT-B,and improper or unexpected hybridization to alleles, with a locus otherthan the normal chromosomal locus for the polynucleotide sequenceencoding PPT-B. The encoded protein may also be "altered" and containdeletions, insertions, or substitutions of amino acid residues whichproduce a silent change and result in a functionally equivalent PPT-B.Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological or immunological activity of PPT-B is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid; positively charged amino acids may include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline, glycine and alanine, asparagine and glutamine, serine andthreonine, and phenylalanine and tyrosine.

"Amino acid sequence" as used herein refers to an oligopeptide, peptide,polypeptide, or protein sequence, and fragment thereof, and to naturallyoccurring or synthetic molecules. Fragments of PPT-B are preferablyabout 5 to about 15 amino acids in length and retain the biologicalactivity or the immunological activity of PPT-B. Where "amino acidsequence" is recited herein to refer to an amino acid sequence of anaturally occurring protein molecule, amino acid sequence, and liketerms, are not meant to limit the amino acid sequence to the complete,native amino acid sequence associated with the recited protein molecule.

"Amplification" as used herein refers to the production of additionalcopies of a nucleic acid sequence and is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art(Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y.).

The term "antagonist" as used herein, refers to a molecule which, whenbound to PPT-B, decreases the amount or the duration of the effect ofthe biological or immunological activity of PPT-B. Antagonists mayinclude antibodies, proteins, nucleic acids, carbohydrates, or any othermolecules which decrease the effect of PPT-B.

As used herein, the term "antibody" refers to intact molecules as wellas fragments thereof, such as Fab F(ab')₂, and Fv, which are capable ofbinding the epitopic determinant. Antibodies that bind PPT-Bpolypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or oligopeptide used to immunize an animal can be derivedfrom the translation of RNA or synthesized chemically and can beconjugated to a carrier protein, if desired. Commonly used carriers thatare chemically coupled to peptides include bovine serum albumin andthyroglobulin, keyhole limpet hemocyanin. The coupled peptide is thenused to immunize the animal (e.g., a mouse, a rat, or a rabbit).

The term "antigenic determinant", as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

The term "antisense", as used herein, refers to any compositioncontaining nucleotide sequences which are complementary to a specificDNA or RNA sequence. The term "antisense strand" is used in reference toa nucleic acid strand that is complementary to the "sense" strand.Antisense molecules include peptide nucleic acids and may be produced byany method including synthesis or transcription. Once introduced into acell, the complementary nucleotides combine with natural sequencesproduced by the cell to form duplexes and block either transcription ortranslation. The designation "negative" is sometimes used in referenceto the antisense strand, and "positive" is sometimes used in referenceto the sense strand.

The term "biologically active", as used herein, refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, "immunologically active" refers to thecapability of the natural, recombinant, or synthetic PPT-B, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

The terms "complementary" or "complementarity", as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. For example, the sequence"A-G-T" binds to the complementary sequence "T-C-A". Complementaritybetween two single-stranded molecules may be "partial", in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands and in thedesign and use of PNA molecules.

A "composition comprising a given polynucleotide sequence" as usedherein refers broadly to any composition containing the givenpolynucleotide sequence. The composition may comprise a dry formulationor an aqueous solution. Compositions comprising polynucleotide sequencesencoding PPT-B (SEQ ID NO:1) or fragments thereof (e.g., SEQ ID NO:2 andfragments thereof) may be employed as hybridization probes. The probesmay be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g. SDS) and other components (e.g., Denhardt's solution,dry milk, salmon sperm DNA. etc.).

"Consensus", as used herein, refers to a nucleic acid sequence which hasbeen resequenced to resolve uncalled bases, has been extended usingXL-PCR (Perkin Elmer, Norwalk, Conn.) in the 5' and/or the 3' directionand resequenced, or has been assembled from the overlapping sequences ofmore than one Incyte Clone using a computer program for fragmentassembly (e.g., GELVIEW fragment assembly system, GCG, Madison, Wis.).Some sequences have been both extended and assembled to produce theconsensus sequence.

The term "correlates with expression of a polynucleotide", as usedherein, indicates that the detection of the presence of ribonucleic acidthat is similar to SEQ ID NO:2 by northern analysis is indicative of thepresence of mRNA encoding PPT-B in a sample and thereby correlates withexpression of the transcript from the polynucleotide encoding theprotein.

A "deletion", as used herein, refers to a change in the amino acid ornucleotide sequence and results in the absence of one or more amino acidresidues or nucleotides.

The term "derivative", as used herein, refers to the chemicalmodification of a nucleic acid encoding or complementary to PPT-B or theencoded PPT-B. Such modifications include, for example, replacement ofhydrogen by an alkyl, acyl, or amino group. A nucleic acid derivativeencodes a polypeptide which retains the biological or immunologicalfunction of the natural molecule. A derivative polypeptide is one whichis modified by glycosylation, pegylation, or any similar process whichretains the biological or immunological function of the polypeptide fromwhich it was derived.

The term "homology", as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence that at leastpartially inhibits an identical sequence from hybridizing to a targetnucleic acid is referred to using the functional term "substantiallyhomologous." The inhibition of hybridization of the completelycomplementary sequence to the target sequence may be examined using ahybridization assay (Southern or northern blot, solution hybridizationand the like) under conditions of low stringency. A substantiallyhomologous sequence or hybridization probe will compete for and inhibitthe binding of a completely homologous sequence to the target sequenceunder conditions of low stringency. This is not to say that conditionsof low stringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e. selective) interaction. The absence ofnon-specific binding may be tested by the use of a second targetsequence which lacks even a partial degree of complementarity (e.g.,less than about 30% identity). In the absence of non-specific binding,the probe will not hybridize to the second non-complementary targetsequence.

Human artificial chromosomes (HACs) are linear microchromosomes whichmay contain DNA sequences of 10K to 10M in size and contain all of theelements required for stable mitotic chromosome segregation andmaintenance (Harrington, J. J. et al. (1997) Nat Genet. 15:345-355).

The term "humanized antibody", as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

The term "hybridization", as used herein, refers to any process by whicha strand of nucleic acid binds with a complementary strand through basepairing.

The term "hybridization complex", as used herein, refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary G and C bases and betweencomplementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀ t or R₀ tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,paper, membranes, filters, chips, pins or glass slides, or any otherappropriate substrate to which cells or their nucleic acids have beenfixed).

An "insertion" or "addition", as used herein, refers to a change in anamino acid or nucleotide sequence resulting in the addition of one ormore amino acid residues or nucleotides, respectively, as compared tothe naturally occurring molecule.

"Microarray" refers to an array of distinct polynucleotides oroligonucleotides synthesized on a substrate, such as paper, nylon orother type of membrane, filter, chip, glass slide, or any other suitablesolid support.

The term "modulate", as used herein, refers to a change in the activityof PPT-B. For example, modulation may cause an increase or a decrease inprotein activity, binding characteristics, or any other biological,functional or immunological properties of PPT-B.

"Nucleic acid sequence" as used herein refers to an oligonucleotide,nucleotide, or polynucleotide, and fragments thereof, and to DNA or RNAof genomic or synthetic origin which may be single- or double-stranded,and represent the sense or antisense strand. "Fragments" are thosenucleic acid sequences which are greater than 60 nucleotides than inlength, and most preferably includes fragments that are at least 100nucleotides or at least 1000 nucleotides, and at least 10,000nucleotides in length.

The term "oligonucleotide" refers to a nucleic acid sequence of at leastabout 6 nucleotides to about 60 nucleotides, preferably about 15 to 30nucleotides, and more preferably about 20 to 25 nucleotides, which canbe used in PCR amplification, hybridization assays, or microassays. Asused herein, oligonucleotide is substantially equivalent to the terms"amplimers", "primers", "oligomers", and "probes", as commonly definedin the art.

"Peptide nucleic acid", PNA as used herein, refers to an antisensemolecule or anti-gene agent which comprises an oligonucleotide of atleast five nucleotides in length linked to a peptide backbone of aminoacid residues which ends in lysine. The terminal lysine conferssolubility to the composition. PNAs may be pegylated to extend theirlifespan in the cell where they preferentially bind complementary singlestranded DNA and RNA and stop transcript elongation (Nielsen, P. E. etal. (1993) Anticancer Drug Des. 8:53-63).

The term "portion", as used herein, with regard to a protein (as in "aportion of a given protein") refers to fragments of that protein. Thefragments may range in size from five amino acid residues to the entireamino acid sequence minus one amino acid. Thus, a protein "comprising atleast a portion of the amino acid sequence of SEQ ID NO:1" encompassesthe full-length PPT-B and fragments thereof.

The term "sample", as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acid encoding PPT-B,or fragments thereof, or PPT-B itself may comprise a bodily fluid,extract from a cell, chromosome, organelle, or membrane isolated from acell, a cell, genomic DNA, RNA, or cDNA(in solution or bound to a solidsupport, a tissue, a tissue print, and the like.

The terms "specific binding" or "specifically binding", as used herein,refers to that interaction between a protein or peptide and an agonist,an antibody and an antagonist. The interaction is dependent upon thepresence of a particular structure (i.e., the antigenic determinant orepitope) of the protein recognized by the binding molecule. For example,if an antibody is specific for epitope "A", the presence of a proteincontaining epitope A (or free, unlabeled A) in a reaction containinglabeled "A" and the antibody will reduce the amount of labeled A boundto the antibody.

The terms "stringent conditions" or "stringency", as used herein, referto the conditions for hybridization as defined by the nucleic acid,salt, and temperature. These conditions are well known in the art andmay be altered in order to identify or detect identical or relatedpolynucleotide sequences. Numerous equivalent conditions comprisingeither low or high stringency depend on factors such as the length andnature of the sequence (DNA, RNA, base composition), nature of thetarget (DNA, RNA, base composition), milieu (in solution or immobilizedon a solid substrate), concentration of salts and other components(e.g., formamide, dextran sulfate and/or polyethylene glycol), andtemperature of the reactions (within a range from about 5° C. below themelting temperature of the probe to about 20° C. to 25° C. below themelting temperature). One or more factors be may be varied to generateconditions of either low or high stringency different from, butequivalent to, the above listed conditions.

The term "substantially purified", as used herein, refers to nucleic oramino acid sequences that are removed from their natural environment,isolated or separated, and are at least 60% free, preferably 75% free,and most preferably 90% free from other components with which they arenaturally associated.

A "substitution", as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

"Transformation", as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the type of host cell beingtransformed and may include, but is not limited to, viral infection,electroporation, heat shock, lipofection, and particle bombardment. Such"transformed" cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as pan of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

A "variant" of PPT-B, as used herein, refers to an amino acid sequencethat is altered by one or more amino acids. The variant may have"conservative" changes, wherein a substituted amino acid has similarstructural or chemical properties, e.g., replacement of leucine withisoleucine. More rarely, a variant may have "nonconservative" changes,e.g., replacement of a glycine with a tryptophan. Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

The Invention

The invention is based on the discovery of a new human preprotachykininB (hereinafter referred to as "PPT-B"), the polynucleotides encodingPPT-B, and the use of these compositions for the diagnosis, prevention,or treatment of neurological disorders and neoplastic disorders.

Nucleic acids encoding the PPT-B of the present invention were firstidentified in Incyte Clone 2109906 from the brain tumor tissue cDNAlibrary (BRAITUT03) using a computer search for amino acid sequencealignments. A consensus sequence, SEQ ID NO:2, was derived from thefollowing overlapping and/or extended nucleic acid sequences: IncyteClones 866703, 866445, 2109906, 2106440 (BRAITUT03), 489306 (HNT2AGT01),2256223 (OVARTUT01), 2207643 (SINTFET03), 1500257, 1498317 (SINTBST01),1384531 and 1380957(BRAITUT08).

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A and 1B.Human PPT-B is 122 amino acids in length and has a potential amidationsite at residue M90; four potential casein kinase II phosphorylationsites at residues S37, S55, T75, S99; one potential protein kinase Cphosphorylation site at residues S37; one potential tyrosine proteinkinase phosphorylation site at residues Y36; and one potential leucinezipper pattern between residues L43 and L64. The hydrophobic amino acidresidues M1 to G20 of human PPT-B are typical of those of a signalpeptide of a secreted molecule. Human PPT-B has three potential sitesfor proteolytic cleavage at K38-R39, K79-R80, and K92-R93 to yield a38-residue peptide homologous to rat preprotachykinin B(50-79) and apeptide with 100% sequence identity to NKB (Helke, C. J. (1990) supra).As shown in FIG. 2, human PPT-B has chemical and structural homologywith bovine PPT-B (GI 163590; SEQ ID NO:3), and rat PPT-B (GI 205724;SEQ ID NO:4). In particular, human PPT-B and bovine PPT-B share 66%identity, human PPT-B and rat PPT-B share 68% identity. As illustratedby FIGS. 3A and 3B, human PPT-B and bovine PPT-B have rather similarhydrophobicity plots. Northern analysis shows the expression of thissequence in various libraries, at least 50% of which are immortalized orcancerous, in particular tumors of the ovary, brain, and adrenal gland;and at least 25% of which involve the sympathetic nervous system.

The invention also encompasses PPT-B variants. A preferred PPT-B variantis one having at least 80%, and more preferably 90%, amino acid sequenceidentity to the PPT-B amino acid sequence (SEQ ID NO:1). A mostpreferred PPT-B variant is one having at least 95% amino acid sequenceidentity to SEQ ID NO:1.

The invention also encompasses polynucleotides which encode PPT-B.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of PPT-B can be used to produce recombinant molecules whichexpress PPT-B. In a particular embodiment, the invention encompasses thepolynucleotide comprising the nucleic acid sequence of SEQ ID NO:2 asshown in FIGS. 1A and 1B.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of nucleotide sequencesencoding PPT-B, some bearing minimal homology to the nucleotidesequences of any known and naturally occurring gene, may be produced.Thus, the invention contemplates each and every possible variation ofnucleotide sequence that could be made by selecting combinations basedon possible codon choices. These combinations are made in accordancewith the standard triplet genetic code as applied to the nucleotidesequence of naturally occurring PPT-B, and all such variations are to beconsidered as being specifically disclosed.

Although nucleotide sequences which encode PPT-B and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring PPT-B under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding PPT-B or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding PPT-B and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

The invention also encompasses production of DNA sequences, or fragmentsthereof, which encode PPT-B and its derivatives, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents that are well known in the art. Moreover, synthetic chemistrymay be used to introduce mutations into a sequence encoding PPT-B or anyfragment thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed nucleotide sequences, and inparticular, those shown in SEQ ID NO:2, under various conditions ofstringency as taught in Wahl, G. M. and S. L. Berger (1987; MethodsEnzymol. 152:399-407) and Kimmel, A. R. (1987; Methods Enzymol.152:507-511).

Methods for DNA sequencing which are well known and generally availablein the art and may be used to practice any of the embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase SEQUENASE (US Biochemical Corp, Cleveland, Ohio), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of polymerases and proofreadingexonucleases such as those found in the ELONGASE amplification systemmarketed by Gibco/BRL (Gaithersburg, Md.). Preferably, the process isautomated with machines such as the MICRO LAB, 2200 (Hamilton, Reno,Nev.), Peltier thermal cycler (PTC200; M J Research, Watertown, Mass.)and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).

The nucleic acid sequences encoding PPT-B may be extended utilizing apartial nucleotide sequence and employing various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. For example, one method which may be employed,"restriction-site" PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to a linker sequence and a primer specific to theknown region. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed usingcommercially available software such as OLIGO 4.06 primer analysissoftware (National Biosciences Inc., Plymouth, Minn.), or anotherappropriate program, to be 22-30 nucleotides in length, to have a GCcontent of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. The method uses several restrictionenzymes to generate a suitable fragment in the known region of a gene.The fragment is then circularized by intramolecular ligation and used asa PCR template.

Another method which may be used is capture PCR which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown fragment of the DNA moleculebefore performing PCR.

Another method which may be used to retrieve unknown sequences is thatof Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PROMOTER FINDERlibraries to walk genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences which contain the 5' regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into 5' non-transcribedregulatory regions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and detection of the emitted wavelengths by a charge coupled devisecamera. Output/light intensity may be converted to electrical signalusing appropriate software (e.g. GENOTYPER and SEQUENCE NAVIGATOR PerkinElmer) and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for the sequencing ofsmall pieces of DNA which might be present in limited amounts in aparticular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode PPT-B may be used in recombinant DNAmolecules to direct expression of PPT-B, fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced, and these sequences may be used to clone and expressPPT-B.

As will be understood by those of skill in the art, it may beadvantageous to produce PPT-B-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce an RNA transcript havingdesirable properties, such as a half-life which is longer than that of atranscript generated from the naturally occurring sequence.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alter PPT-Bencoding sequences for a variety of reasons, including but not limitedto, alterations which modify the cloning, processing, and/or expressionof the gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, introduce mutations, and so forth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding PPT-B may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of PPT-B activity, it may be useful toencode a chimeric PPT-B protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the PPT-B encoding sequence and theheterologous protein sequence, so that PPT-B may be cleaved and purifiedaway from the heterologous moiety.

In another embodiment, sequences encoding PPT-B may be synthesized, inwhole or in part, using chemical methods well known in the art (seeCaruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223,Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of PPT-B, or a fragment thereof.For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge, J. Y. et al. (1995) Science269:202-204) and automated synthesis may be achieved, for example, usingthe ABI 431A Peptide Synthesizer (Perkin Elmer).

The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, W H Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of PPT-B, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

In order to express a biologically active PPT-B, the nucleotidesequences encoding PPT-B or functional equivalents, may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding PPT-B andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

A variety of expression vector/host systems may be utilized to containand express sequences encoding PPT-B. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

The "control elements" or "regulatory sequences" are thosenon-translated regions of the vector--enhancers, promoters, 5' and 3'untranslated regions--which interact with host cellular proteins tocarry out transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene,LaJolla, Calif.) or PSPORT1 plasmid (Gibco BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding PPT-B,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for PPT-B. For example, when largequantities of PPT-B are needed for the induction of antibodies, vectorswhich direct high level expression of fusion proteins that are readilypurified may be used. Such vectors include, but are not limited to, themultifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene), in which the sequence encoding PPT-B may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al. (1987) Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression ofsequences encoding PPT-B may be driven by any of a number of promoters.For example, viral promoters such as the 35S and 19S promoters of CaMVmay be used alone or in combination with the omega leader sequence fromTMV (Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plantpromoters such as the small subunit of RUBISCO or heat shock promotersmay be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R.et al. 30 (1984) Science 224:838-843; and Winter, J. et al. (1991)Results Probl. Cell Differ. 17:85-105). These constructs can beintroduced into plant cells by direct DNA transformation orpathogen-mediated transfection. Such techniques are described in anumber of generally available reviews (see, for example, Hobbs, S. orMurry, L. E. in McGraw Hill Yearbook of Science and Technology (1992)McGraw Hill, New York. N.Y.; pp. 191-196.

An insect system may also be used to express PPT-B. For example, in onesuch system, Autographa californica nuclear polyhedrosis virus (AcNPV)is used as a vector to express foreign genes in Spodoptera frugiperdacells or in Trichoplusia larvae. The sequences encoding PPT-B may becloned into a non-essential region of the virus, such as the polyhedringene, and placed under control of the polyhedrin promoter. Successfulinsertion of PPT-B will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which PPT-B may be expressed (Engelhard, E. K. et al. (1994)Proc. Nat. Acad. Sci. 91:3224-3227).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding PPT-B may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing PPT-B in infected host cells (Logan, J. and Shenk,T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained and expressed in aplasmid. HACs of 6 to 10M are constructed and delivered via conventionaldelivery methods (liposomes, polycationic amino polymers, or vesicles)for therapeutic purposes.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding PPT-B. Such signals include the ATGinitiation codon and adjacent sequences. In cases where sequencesencoding PPT-B, its initiation codon, and upstream sequences areinserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a fragment thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et al. (1994) Results Probl. Cell Differ.20:125-162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a "prepro" form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and WI38), are available from the American TypeCulture Collection (ATCC; Bethesda, Md.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressPPT-B may be transformed using expression vectors which may containviral origins of replication and/or endogenous expression elements and aselectable marker gene on the same or on a separate vector. Followingthe introduction of the vector, cells may be allowed to grow for 1-2days in an enriched media before they are switched to selective media.The purpose of the selectable marker is to confer resistance toselection, and its presence allows growth and recovery of cells whichsuccessfully express the introduced sequences. Resistant clones ofstably transformed cells may be proliferated using tissue culturetechniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) geneswhich can be employed in tk⁻ or aprt⁻ cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr, which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14); and als or pat, which confer resistance to chlorsulfnron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding PPT-B isinserted within a marker gene sequence, transformed cells containingsequences encoding PPT-B can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding PPT-B under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

Alternatively, host cells which contain the nucleic acid sequenceencoding PPT-B and express PPT-B may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

The presence of polynucleotide sequences encoding PPT-B can be detectedby DNA-DNA or DNA-RNA hybridization or amplification using probes orfragments or fragments of polynucleotides encoding PPT-B. Nucleic acidamplification based assays involve the use of oligonucleotides oroligomers based on the sequences encoding PPT-B to detect transformantscontaining DNA or RNA encoding PPT-B.

A variety of protocols for detecting and measuring the expression ofPPT-B, using either polyclonal or monoclonal antibodies specific for theprotein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson PPT-B is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding PPT-B includeoligolabeling, nick translation, end-labeling or PCR amplification usinga labeled nucleotide. Alternatively, the sequences encoding PPT-B, orany fragments thereof may be cloned into a vector for the production ofan mRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.);Promega (Madison Wis.); and U.S. Biochemical Corp., Cleveland, Ohio).Suitable reporter molecules or labels, which may be used for ease ofdetection, include radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding PPT-B may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodePPT-B may be designed to contain signal sequences which direct secretionof PPT-B through a prokaryotic or eukaryotic cell membrane. Otherconstructions may be used to join sequences encoding PPT-B to nucleotidesequence encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and PPT-B may be used to facilitate purification. One suchexpression vector provides for expression of a fusion protein containingPPT-B and a nucleic acid encoding 6 histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification on IMIAC (immobilized metal ion affinitychromatography, as described in Porath, J. et al. (1992, Prot. Exp.Purif. 3:263-281) while the enterokinase cleavage site provides a meansfor purifying PPT-B from the fusion protein. A discussion of vectorswhich contain fusion proteins is provided in Kroll, D. J. et al. (1993;DNA Cell Biol. 12:441-453).

In addition to recombinant production, fragments of PPT-B may beproduced by direct peptide synthesis using solid-phase techniquesMerrifield J. (1963) J. Arn. Chem. Soc. 85:2149-2154). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using an Applied Biosystems 431APeptide Synthesizer (Perkin Elmer). Various fragments of PPT-B may bechemically synthesized separately and combined using chemical methods toproduce the full length molecule.

Therapeutics

Chemical and structural homology exits among human PPT-B, bovine PPT-B(GI 163590), and rat PPT-B (GI 205725). In addition, PPT-B is expressedin neural tissue, gastrointestinal tissue, adrenal tissue, proliferatingtissue, and rapidly dividing cells. Therefore, PPT-B appears to play arole in neurological disorders and neoplastic disorders, particularlydisorders in which PPT-B is overexpressed, or associated with regulationby the sympathetic nervous system.

Therefore, in one embodiment, PPT-B or a fragment or derivative thereofmay be administered to a subject to treat a neurological disorder. Suchdisorders include, but are not limited to, akathesia, Alzheimer'sdisease, amnesia, amyotrophic lateral sclerosis, bipolar disorder,catatonia, cerebral neoplasms, dementia, depression, Down's syndrome,tardive dyskinesia, dystonias, epilepsy, Huntington's disease, multiplesclerosis, neurofibromatosis, Parkinson's disease, paranoid psychoses,schizophrenia, and Tourette's disorder; angina, anaphylactic shock,arrhythmias, asthma, cardiovascular shock, Cushing's syndrome,hypertension, hypoglycemia, myocardial infarction, migraine, andpheochromocytoma.

In another embodiment, a vector capable of expressing PPT-B, or afragment or a derivative thereof, may also be administered to a subjectto treat a neurological disorder, including, but not limited to, thoselisted above.

In still another embodiment, an agonist of PPT-B may also beadministered to a subject to treat a neurological disorder, including,but not limited to, those listed above.

In one embodiment, an antagonist of PPT-B may be administered to asubject to prevent or treat a neoplastic disorder. Such disorders mayinclude, but are not limited to, adenocarcinoma, leukemia, lymphoma,melanoma, myeloma, sarcoma, and teratocarcinoma, and particularlycancers of the adrenal gland, bladder, bone, bone marrow, brain, breast,cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney,liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate,salivary glands, skin, spleen, testis, thymus, thyroid, and uterus. Inone aspect, antibodies which specifically bind PPT-B may be useddirectly as an antagonist or indirectly as a targeting or deliverymechanism for bringing a pharmaceutical agent to cells or tissue whichexpress PPT-B.

In another embodiment, a vector expressing the complement of thepolynucleotide encoding PPT-B may be administered to a subject to treator prevent a neoplastic disorder including, but not limited to, thoselisted above.

In other embodiments, any of the proteins, antagonists, antibodies,agonists, complementary sequences or vectors of the invention may beadministered in combination with other appropriate therapeutic agents.Selection of the appropriate agents for use in combination therapy maybe made by one of ordinary skill in the art, according to conventionalpharmaceutical principles. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

Antagonists or inhibitors of PPT-B may be produced using methods whichare generally known in the art. In particular, purified PPT-B may beused to produce antibodies or to screen libraries of pharmaceuticalagents to identify those which specifically bind PPT-B.

Antibodies to PPT-B may be generated using methods that are well knownin the art. Such antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric, single chain, Fab fragments, andfragments produced by a Fab expression library. Neutralizing antibodies,(i.e., those which inhibit dimer formation) are especially preferred fortherapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith PPT-B or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corvnebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to PPT-B have an amino acid sequence consisting of atleast five amino acids and more preferably at least 10 amino acids. Itis also preferable that they are identical to a portion of the aminoacid sequence of the natural protein, and they may contain the entireamino acid sequence of a small, naturally occurring molecule. Shortstretches of PPT-B amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule.

Monoclonal antibodies to PPT-B may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. etal. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc.Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al. (1984) Mol. Cell Biol.62:109-120).

In addition, techniques developed for the production of "chimericantibodies", the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to producePPT-B-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobulin libraries(Burton D. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature(Orlandi, R. et al. (1989) Proc. Nati. Acad. Sci. 86:3833-3837; Winter,G. et al. (1991) Nature 349:293-299).

Antibody fragments which contain specific binding sites for PPT-B mayalso be generated. For example, such fragments include, but are notlimited to, the F(ab')2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab')2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between PPT-B and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering PPT-B epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra).

In another embodiment of the invention, the polynucleotides encodingPPT-B, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect, the complement of thepolynucleotide encoding PPT-B may be used in situations in which itwould be desirable to block the transcription of the mRNA. Inparticular, cells may be transformed with sequences complementary topolynucleotides encoding PPT-B. Thus, complementary molecules orfragments may be used to modulate PPT-B activity, or to achieveregulation of gene function. Such technology is now well known in theart, and sense or antisense oligonucleotides or larger fragments, can bedesigned from various locations along the coding or control regions ofsequences encoding PPT-B.

Expression vectors derived from retro viruses, adenovirus, herpes orvaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors which will express nucleic acid sequencewhich is complementary to the polynucleotides of the gene encodingPPT-B. These techniques are described both in Sambrook et al. (supra)and in Ausubel et al. (supra).

Genes encoding PPT-B can be turned off by transforming a cell or tissuewith expression vectors which express high levels of a polynucleotide orfragment thereof which encodes PPT-B. Such constructs may be used tointroduce untranslatable sense or antisense sequences into a cell. Evenin the absence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector and even longer if appropriate replicationelements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning complementary sequences or antisense molecules (DNA, RNA, orPNA) to the control, 5' or regulatory regions of the gene encoding PPT-B(signal sequence, promoters, enhancers, and introns). Oligonucleotidesderived from the transcription initiation site, e.g., between positions-10 and +10 from the start site, are preferred. Similarly, inhibitioncan be achieved using "triple helix" base-pairing methodology. Triplehelix pairing is useful because it causes inhibition of the ability ofthe double helix to open sufficiently for the binding of polymerases,transcription factors, or regulatory molecules. Recent therapeuticadvances using triplex DNA have been described in the literature (Gee,J. E. et al. (1994) In: Huber, B. E. and B. I. Carr, Molecular andImmunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.). Thecomplementary sequence or antisense molecule may also be designed toblock translation of mRNA by preventing the transcript from binding toribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding PPT-B.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the inventionmay be prepared by any method known in the art for the synthesis ofnucleic acid molecules. These include techniques for chemicallysynthesizing oligonucleotides such as solid phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding PPT-B. SuchDNA sequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,these cDNA constructs that synthesize complementary RNA constitutivelyor inducibly can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5' and/or 3' ends of the moleculeor the use of phosphorothioate or 2' O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections orpolycationic amino polymers (Goldman, C. K. et al. (1997) NatureBiotechnology 15:462-66; incorporated herein by reference) may beachieved using methods which are well known in the art.

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, is monkeys, and most preferably,humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition, in conjunction with a pharmaceuticallyacceptable carrier, for any of the therapeutic effects discussed above.Such pharmaceutical compositions may consist of PPT-B, antibodies toPPT-B, mimetics, agonists, antagonists, or inhibitors of PPT-B. Thecompositions may be administered alone or in combination with at leastone other agent, such as stabilizing compound, which may be administeredin any sterile, biocompatible pharmaceutical carrier, including, but notlimited to, saline, buffered saline, dextrose, and water. Thecompositions may be administered to a patient alone, or in combinationwith other agents, drugs or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Non-lipid polycationicamino polymers may also be used for delivery. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic acids etc. Saltstend to be more soluble in aqueous or other protonic solvents than arethe corresponding free base forms. In other cases, the preferredpreparation may be a lyophilized powder which may contain any or all ofthe following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, ata pH range of 4.5 to 5.5, that is combined with buffer prior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of PPT-B, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually mice, rabbits, dogs, or pigs. The animal modelmay also be used to determine the appropriate concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example PPT-B or fragments thereof, antibodies of PPT-B,agonists, antagonists or inhibitors of PPT-B, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio of or to therapeutic effects is thetherapeutic index, which can be expressed as the ratio LD50/ED50.

Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly. deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

Diagnostics

In another embodiment, antibodies which specifically bind PPT-B may beused for the diagnosis of conditions or diseases characterized byexpression of PPT-B, or in assays to monitor patients being treated withPPT-B, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for PPT-B includemethods which utilize the antibody and a label to detect PPT-B in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

A variety of protocols including ELISA, RIA, and FACS for measuringPPT-B are known in the art and provide a basis for diagnosing altered orabnormal levels of PPT-B expression. Normal or standard values for PPT-Bexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toPPT-B under conditions suitable for complex formation. The amount ofstandard complex formation may be quantified by various methods, butpreferably by photometric, means. Quantities of PPT-B expressed insubject samples from biopsied tissues are compared with the standardvalues. Deviation between standard and subject values establishes theparameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingPPT-B may be used for diagnostic purposes. The polynucleotides which maybe used include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofPPT-B may be correlated with disease. The diagnostic assay may be usedto distinguish between absence, presence, and excess expression ofPPT-B, and to monitor regulation of PPT-B levels during therapeuticintervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding PPT-B or closely related molecules, may be used to identifynucleic acid sequences which encode PPT-B. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5' regulatory region, or a less specific region,e.g., especially in the 3' coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding PPT-B, alleles, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably contain at least 50% of the nucleotides from any ofthe PPT-B encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID NO:2 or from genomic sequence including promoter, enhancerelements, and introns of the naturally occurring PPT-B.

Means for producing specific hybridization probes for DNAs encodingPPT-B include the cloning of nucleic acid sequences encoding PPT-B orPPT-B derivatives into vectors for the production of mRNA probes. Suchvectors are known in the art, commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

Polynucleotide sequences encoding PPT-B may be used for the diagnosis ofconditions, disorders, or diseases which are associated with expressionof PPT-B. Examples of such conditions or diseases include, but are notlimited to, akathesia, Alzheimer's disease, amnesia, amyotrophic lateralsclerosis, bipolar disorder, catatonia, cerebral neoplasms, dementia,depression, Down's syndrome, tardive dyskinesia, dystonias, epilepsy,Huntington's disease, multiple sclerosis, neurofibromatosis, Parkinson'sdisease, paranoid psychoses, schizophrenia, and Tourette's disorder;angina, anaphylactic shock, arrhythmias, asthma, cardiovascular shock,Cushing's syndrome, hypertension, hypoglycemia, myocardial infarction,migraine, and pheochromocytoma; adenocarcinoma, leukemia, lymphoma,melanoma, myeloma, sarcoma, and teratocarcinoma, and particularlycancers of the adrenal gland, bladder, bone, bone marrow, brain, breast,cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney,liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate,salivary glands, skin, spleen, testis, thymus, thyroid, and uterus. Thepolynucleotide sequences encoding PPT-B may be used in Southern ornorthern analysis, dot blot, or other membrane-based technologies; inPCR technologies; or in dipstick, pin, ELISA assays or microarraysutilizing fluids or tissues from patient biopsies to detect alteredPPT-B expression. Such qualitative or quantitative methods are wellknown in the art.

In a particular aspect, the nucleotide sequences encoding PPT-B may beuseful in assays that detect activation or induction of various cancers,particularly those mentioned above. The nucleotide sequences encodingPPT-B may be labeled by standard methods, and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the biopsied or extracted sample issignificantly altered from that of a comparable control sample, thenucleotide sequences have hybridized with nucleotide sequences in thesample, and the presence of altered levels of nucleotide sequencesencoding PPT-B in the sample indicates the presence of the associateddisease. Such assays may also be used to evaluate the efficacy of aparticular therapeutic treatment regimen in animal studies, in clinicaltrials, or in monitoring the treatment of an individual patient.

In order to provide a basis for the diagnosis of disease associated withexpression of PPT-B, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, which encodes PPT-B, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withthose from an experiment where a known amount of a substantiallypurified polynucleotide is used. Standard values obtained from normalsamples may be compared with values obtained from samples from patientswho are symptomatic for disease. Deviation between standard and subjectvalues is used to establish the presence of disease.

Once disease is established and a treatment protocol is initiated,hybridization assays may be repeated on a regular basis to evaluatewhether the level of expression in the patient begins to approximatethat which is observed in the normal patient. The results obtained fromsuccessive assays may be used to show the efficacy of treatment over aperiod ranging from several days to months.

With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding PPT-B may involve the use of PCR. Such oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably consist of two nucleotide sequences,one with sense orientation (5'→3') and another with antisense (3'←5'),employed under optimized conditions for identification of a specificgene or condition. The same two oligomers, nested sets of oligomers, oreven a degenerate pool of oligomers may be employed under less stringentconditions for detection and/or quantitation of closely related DNA orRNA sequences.

Methods which may also be used to quantitate the expression of PPT-Binclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and standard curves onto which the experimentalresults are interpolated (Melby, P. C. et al. (1993) J. Immunol.Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem.212:229-236). The speed of quantitation of multiple samples may beaccelerated by running the assay in an ELISA format where the oligomerof interest is presented in various dilutions and a spectrophotometricor calorimetric response gives rapid quantitation.

In further embodiments, oligonucleotides derived from any of thepolynucleotide sequences described herein may be used as targets inmicroarrays. The microarrays can be used to monitor the expression levelof large numbers of genes simultaneously (to produce a transcriptimage), and to identify genetic variants, mutations and polymorphisms.This information will be useful in determining gene function,understanding the genetic basis of disease, diagnosing disease, and indeveloping and monitoring the activity of therapeutic agents.

In one embodiment, the microarray is prepared and used according to themethods described in PCT application WO95/11995 (Chee et al.), Lockhart,D. J. et al. (1996; Nat. Biotech. 14:1675-1680) and Schena, M. et al.(1996; Proc. Natl. Acad. Sci. 93:10614-10619), all of which areincorporated herein in their entirety by reference.

The microarray is preferably composed of a large number of unique,single-stranded nucleic acid sequences, usually either syntheticantisense oligonucleotides or fragments of cDNAs fixed to a solidsupport. Microarrays may contain oligonucleotides which cover the known5', or 3', sequence, or contain sequential oligonucleotides which coverthe full length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray may be oligonucleotides that are specific to a gene orgenes of interest in which at least a fragment of the sequence is knownor that are specific to one or more unidentified cDNAs which are commonto a particular cell type, developmental or disease state.

In order to produce oligonucleotides to a known sequence for amicroarray, the gene of interest is examined using a computer algorithmwhich starts at the 5' or more preferably at the 3' end of thenucleotide sequence. The algorithm identifies oligomers of definedlength that are unique to the gene, have a GC content within a rangesuitable for hybridization, and lack predicted secondary structure thatmay interfere with hybridization. The oligomers are synthesized atdesignated areas on a substrate using a light-directed chemical process.The substrate may be paper, nylon or other type of membrane, filter,chip, glass slide or any ether suitable solid support.

In another aspect, the oligonucleotides may be synthesized on thesurface of the substrate by using a chemical coupling procedure and anink jet application apparatus, as described in PCT application WO95/251116 (Baldeschweiler et al.) which is incorporated herein in itsentirety by reference. In another aspect, a "gridded" array analogous toa dot (or slot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array may beproduced by hand or using available devises (slot blot or dot blotapparatus) materials and machines (including robotic instruments) andcontain grids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144dots, or any other multiple which lends itself to the efficient use ofcommercially available instrumentation.

In order to conduct sample analysis using the microarrays, the RNA orDNA from a biological sample is made into hybridization probes. The mRNAis isolated, and cDNA is produced and used as a template to makeantisense RNA (aRNA). The aRNA is amplified in the presence offluorescent nucleotides, and labeled probes are incubated with themicroarray so that the probe sequences hybridize to complementaryoligonucleotides of the microarray. Incubation conditions are adjustedso that hybridization occurs with precise complementary matches or withvarious degrees of less complementarity. After removal of nonhybridizedprobes, a scanner is used to determine the levels and patterns offluorescence. The scanned images are examined to determine degree ofcomplementarity and the relative abundance of each oligonucleotidesequence on the microarray. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large scale correlationstudies on the sequences, mutations, variants, or polymorphisms amongsamples.

In another embodiment of the invention, the nucleic acid sequences whichencode PPT-B may also be used to generate hybridization probes which areuseful for mapping the naturally occurring genomic sequence. Thesequences may be mapped to a particular chromosome, to a specific regionof a chromosome or to artificial chromosome constructions, such as humanartificial chromosomes (HACs), yeast artificial chromosomes (YACs),bacterial artificial chromosomes (BACs), bacterial P1 constructions orsingle chromosome cDNA libraries as reviewed in Price, C. M. (1993)Blood Rev. 7:127-134, and Trask, B. J. (1991) Trends Genet. 7:149-154.

Fluorescent in situ hybridization (FISH as described in Verma et al.(1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press,New York, N.Y.) may be correlated with other physical chromosome mappingtechniques and genetic map data. Examples of genetic map data can befound in various scientific journals or at Online Mendelian Inheritancein Man (OMIM). Correlation between the location of the gene encodingPPT-B on a physical chromosomal map and a specific disease, orpredisposition to a specific disease, may help delimit the region of DNAassociated with that genetic disease. The nucleotide sequences of thesubject invention may be used to detect differences in gene sequencesbetween normal, carrier, or affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques such as linkage analysis using established chromosomalmarkers may be used for extending genetic maps. Often the placement of agene on the chromosome of another mammalian species, such as mouse, mayreveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms, or parts thereof, by physical mapping. This providesvaluable information to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, for example, AT to 11q22-23 (Gatti, R. A. etal. (1988) Nature 336:577-580), any sequences mapping to that area mayrepresent associated or regulatory genes for further investigation. Thenucleotide sequence of the subject invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,etc. among normal, carrier, or affected individuals.

In another embodiment of the invention, PPT-B, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweenPPT-B and the agent being tested, may be measured.

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe protein of interest as described in published PCT applicationWO84/03564. In this method, as applied to PPT-B large numbers ofdifferent small test compounds are synthesized on a solid substrate,such as plastic pins or some other surface. The test compounds arereacted with PPT-B, or fragments thereof, and washed. Bound PPT-B isthen detected by methods well known in the art. Purified PPT-B can alsobe coated directly onto plates for use in the aforementioned drugscreening techniques. Alternatively, non-neutralizing antibodies can beused to capture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding PPT-B specificallycompete with a test compound for binding PPT-B. In this manner, theantibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with PPT-B.

In additional embodiments, the nucleotide sequences which encode PPT-Bmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES

I BRAITUT03 cDNA Library Construction

The BRAITUT03 cDNA library was constructed from astrocytoma tissue (leftfrontal lobe) which was obtained from a 17-year-old Caucasian female byexcision of cerebral meningeal lesion. The pathology report indicated agrade IV fibrillary giant and small cell astrocytoma. The patient had ahistory of benign hypertension. Dexamethasone (Merck & Co., West Point,Pa.) was given to reduce inflammation of brain tissue.

The frozen tissue was homogenized and lysed using a Polytron-PT 3000homogenizer (Brinkmann Instruments, Inc. Westbury, N.Y.) in guanidiniumisothiocyanate solution. The lysates were extracted once with acidphenol at pH 4.0 per Stratagene's RNA isolation protocol (StratageneInc, San Diego, Calif.). The RNA was extracted twice with an equalvolume of acid phenol, reprecipitated using 0.3 M sodium acetate and 2.5volumes of ethanol, resuspended in DEPC-treated water and DNase treatedfor 25 min at 37° C. mRNAs were isolated using the OLIGOTEX kit (QIAGENInc, Chatsworth, Calif.) and used to construct the cDNA library.

The RNA was handled according to the recommended protocols in theSUPERSCRIPT plasmid system for cDNA synthesis and plasmid cloning (Cat.#18248-013; Gibco/BRL, Gaithersburg, Md.). cDNAs were fractionated on aSEPHAROSE CL4B column (Cat. #275105, Pharmacia. Alameda, Calif.), andthose cDNAs exceeding 400 bp were ligated into PSPORT1. The plasmidPSPORT1 was subsequently transformed into DH5α competent cells (Cat.#18258-012, Gibco/BRL, Gaithersburg, Md.).

II Isolation and Sequencing of cDNA Clones

Plasmid DNA was released from the cells and purified using the R.E.A.L.PREP 96 plasmid kit (Catalog #26173; QIAGEN, Inc). The recommendedprotocol was employed except for the following changes: 1) the bacteriawere cultured in 1 ml of sterile Terrific Broth (Catalog #22711,Gibco/BRL) with carbenicillin at 25 mg/L and glycerol at 0.4%; 2) thecultures were incubated for 19 hours after the wells were inoculated andthen lysed with 0.3 ml of lysis buffer; 3) following isopropanolprecipitation, the plasmid DNA pellet was resuspended in 0.1 ml ofdistilled water. After the last step in the protocol, samples weretransferred to a Beckman 96-well block for storage at 4° C.

The cDNAs were sequenced by the method of Sanger F. and A. R. Coulson(1975; J. Mol. Biol. 94:441f), using a MICROLAB 2200 (Hamilton, Reno,Nev.) in combination with Peltier thermal cyclers (PTC200 from MJResearch, Watertown, Mass.) and Applied Biosystems 377 DNA sequencingsystems; and the reading frame was determined.

III Homology Searching of cDNA Clones and Their Deduced Proteins

The nucleotide sequences of the Sequence Listing or amino acid sequencesdeduced from them were used as query sequences against databases such asGenBank, SwissProt, BLOCKS, and Pima II. These databases which containpreviously identified and annotated sequences were searched for regionsof homology (similarity) using BLAST, which stands for Basic LocalAlignment Search Tool (Altschul, S. F. (1993) J. Mol. Evol. 36:290-300;Altschul et al. (1990) J. Mol. Biol. 215:403-410).

BLAST produces alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST is especially useful in determining exact matches orin identifying homologs which may be of prokaryotic (bacterial) oreukaryotic (animal, fungal or plant) origin. Other algorithms such asthe one described in Smith R. F. and T. F. Smith (1992; ProteinEngineering 5:35-51), incorporated herein by reference, can be used whendealing with primary sequence patterns and secondary structure gappenalties. As disclosed in this application, the sequences have lengthsof at least 49 nucleotides, and no more than 12% uncalled bases (where Nis recorded rather than A, C, G, or T).

The BLAST approach, as detailed in Karlin, S. and S. F. Atschul (1993;Proc. Nat. Acad. Sci. 90:5873-7) and incorporated herein by reference,searches for matches between a query sequence and a database sequence,to evaluate the statistical significance of any matches found, and toreport only those matches which satisfy the user-selected threshold ofsignificance. In this application, threshold was set at 10⁻²⁵ fornucleotides and 10⁻¹⁴ for peptides.

Incyte nucleotide sequences were searched against the GenBank databasesfor primate (pri), rodent (rod), and mammalian sequences (mam), anddeduced amino acid sequences from the same clones are searched againstGenBank functional protein databases, mammalian (mamp), vertebrate(vrtp) and eukaryote (eukp), for homology. The relevant database for aparticular match were reported as a GIxxx±p (where xxx is pri, rod, etcand if present, p=peptide).

IV Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound (Sambrook et al., supra).

Analogous computer techniques using BLAST (Altschul, S. F. 1993 and1990, supra) are used to search for identical or related molecules innucleotide databases such as GenBank or the LIFESEQ database (IncytePharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

The basis of the search is the product score which is defined as:##EQU1## The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1-2% error; and at 70, the match will be exact. Homologous moleculesare usually identified by selecting those which show product scoresbetween 15 and 40, although lower scores may identify related molecules.

The results of northern analysis are reported as a list of libraries inwhich the transcript encoding PPT-B occurs. Abundance and percentabundance are also reported. Abundance directly reflects the number oftimes a particular transcript is represented in a cDNA library, andpercent abundance is abundance divided by the total number of sequencesexamined in the cDNA library.

V Extension of PPT-B Encoding Polynucleotides

The nucleic acid sequence of the Incyte Clone 2109906 was used to designoligonucleotide primers for extending a partial nucleotide sequence tofull length. One primer was synthesized to initiate extension in theantisense direction, and the other was synthesized to extend sequence inthe sense direction. Primers were used to facilitate the extension ofthe known sequence "outward" generating amplicons containing new,unknown nucleotide sequence for the region of interest. The initialprimers were designed from the cDNA using OLIGO 4.06 software (NationalBiosciences), or another appropriate program, to be about 22 to about 30nucleotides in length, to have a GC content of 50% or more, and toanneal to the target sequence at temperatures of about 68° to about 72°C. Any stretch of nucleotides which would result in hairpin structuresand primer-primer dimerizations was avoided.

Selected human cDNA libraries (Gibco/BRL) were used to extend thesequence If more than one extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

High fidelity amplification was obtained by following the instructionsfor the XL-PCR kit (Perkin Elmer) and thoroughly mixing the enzyme andreaction mix. Beginning with 40 pmol of each primer and the recommendedconcentrations of all other components of the kit, PCR was performedusing the Peltier thermal cycler (PTC200; M. J. Research, Watertown,Mass.) and the following parameters:

    ______________________________________                                        Step 1       94° C. for 1 min (initial denaturation)                   Step 2       65° C. for 1 min                                          Step 3       68° C. for 6 min                                          Step 4       94° C. for 15 sec                                         Step 5       65° C. for 1 min                                          Step 6       68° C. for 7 min                                          Step 7       Repeat step 4-6 for 15 additional cycles                         Step 8       94° C. for 15 sec                                         Step 9       65° C. for 1 min                                          Step 10      68° C. for 7:15 min                                       Step 11      Repeat step 8-10 for 12 cycles                                   Step 12      72° C. for 8 min                                          Step 13      4° C. (and holding)                                       ______________________________________                                    

A 5-10 μaliquot of the reaction mixture was analyzed by electrophoresison a low concentration (about 0.6-0.8%) agarose mini-gel to determinewhich reactions were successful in extending the sequence. Bands thoughtto contain the largest products were excised from the gel, purifiedusing QIAQUICK (QIAGEN Inc., Chatsworth, Calif.), and trimmed ofoverhangs using Klenow enzyme to facilitate religation and cloning.

After ethanol precipitation, the products were redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) were transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the E. coli mixture was platedon Luria Bertani (LB)-agar (Sambrook et al., supra) containing 2× Carb.The following day, several colonies were randomly picked from each plateand cultured in 150 μl of liquid LB/2× Carb medium placed in anindividual well of an appropriate, commercially-available, sterile96-well microtiter plate. The following day, 5 μl of each overnightculture was transferred into a non-sterile 96-well plate and afterdilution 1:10 with water, 5 μl of each sample was transferred into a PCRarray.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer, and one orboth of the gene specific primers used for the extension reaction wereadded to each well. Amplification was performed using the followingconditions:

    ______________________________________                                        Step 1     94° C. for 60 sec                                           Step 2     94° C. for 20 sec                                           Step 3     55° C. for 30 sec                                           Step 4     72° C. for 90 sec                                           Step 5     Repeat steps 2-4 for an additional 29 cycles                       Step 6     72° C. for 180 sec                                          Step 7     4° C. (and holding)                                         ______________________________________                                    

Aliquots of the PCR reactions were run on agarose gels together withmolecular weight markers. The sizes of the PCR products were compared tothe original partial cDNAs, and appropriate clones were selected,ligated into plasmid, and sequenced.

In like manner, the nucleotide sequence of SEQ ID NO:2 is used to obtain5' regulatory sequences using the procedure above, oligonucleotidesdesigned for 5' extension, and an appropriate genomic library.

VI Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:2 are employed to screencDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 (National Biosciences), labeled by combining 50 pmolof each oligomer and 250 μCi of [γ³² P] adenosine triphosphate(Amersham) and T4 polynucleotide kinase (DuPont NEN, Boston, Mass.). Thelabeled oligonucleotides are substantially purified with Sephadex G-25superfine resin column (Pharmacia & Upjohn). A aliquot containing 10⁷counts per minute of the labeled probe is used in a typicalmembrane-based hybridization analysis of human genomic DNA digested withone of the following endonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1,or Pvu II; DuPont NEN).

The DNA from each digest is fractionated on a 0.7 percent agarose geland transferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film(Kodak, Rochester, N.Y.) is exposed to the blots in a Phosphorimagercassette (Molecular Dynamics, Sunnyvale, Calif.) hybridization patternsare compared visually.

VII Microarrays

To produce oligonucleotides for a microarray, the nucleotide sequencedescribed herein is examined using a computer algorithm which starts atthe 3' end of the nucleotide sequence. The algorithm identifiesoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that would interfere with hybridization. Thealgorithm identifies 20 sequence-specific oligonucleotides of 20nucleotides in length (20-mers). A matched set of oligonucleotides iscreated in which one nucleotide in the center of each sequence isaltered. This process is repeated for each gene in the microarray, anddouble sets of twenty 20 mers are synthesized and arranged on thesurface of the silicon chip using a light-directed chemical process(Chee, M. et al., PCT/WO95/11995, incorporated herein by reference).

In the alternative, a chemical coupling procedure and an ink jet deviceare used to synthesize oligomers on the surface of a substrate(Baldeschweiler, J. D. et al., PCT/WO95/25 116, incorporated herein byreference). In another alternative, a "gridded" array analogous to a dot(or slot) blot is used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array may beproduced by hand or using available materials and machines and containgrids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144 dots.After hybridization, the microarray is washed to remove nonhybridizedprobes, and a scanner is used to determine the levels and patterns offluorescence. The scanned images are examined to determine degree ofcomplementarity and the relative abundance of each oligonucleotidesequence on the micro-array.

VIII Complementary Polynucleotides

Sequence complementary to the PPT-B-encoding sequence, or any partthereof, is used to decrease or inhibit expression of naturallyoccurring PPT-B. Although use of oligonucleotides comprising from about15 to about 30 base-pairs is described, essentially the same procedureis used with smaller or larger sequence fragments. Appropriateoligonucleotides are designed using OLIGO 4.06 software and the codingsequence of PPT-B, SEQ ID NO:1. To inhibit transcription, acomplementary oligonucleotide is designed from the most unique 5'sequence and used to prevent promoter binding to the coding sequence. Toinhibit translation, a complementary oligonucleotide is designed toprevent ribosomal binding to the PPT-B-encoding transcript.

IX Expression of PPT-B

Expression of PPT-B is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector is also used to express PPT-B in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terninal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

Induction of an isolated, transformed bacterial strain with IPTG usingstandard methods produces a fusion protein which consists of the firsteight residues of β-galactosidase, about 5 to 15 residues of linker, andthe full length protein. The signal residues direct the secretion ofPPT-B into the bacterial growth media which can be used directly in thefollowing assay for activity.

X Demonstration of PPT-B Activity

The assay is based on the increase in firefly luciferase activity in thereporter cell line A20/NK3-17/3, that has been stably transfected withthe cDNA for the human neurokinin receptor NK-3, following treatmentwith the proteolytic product of PPT-B, NKB (Stratowa, C. et al. (1995)J. Recept. Signal Transduct. Res. 15:617-630). Luciferase activity isinduced by hydrolysis of endogenous phosphatidylinositol by thephospholipase C- NK-3 receptor complex. About 10,000 cells/well areseeded into light-impermeable 96-well microtiter plates MICROLITE,Dynatech Laboratories, Burlington, Mass.) and incubated for 18 hours at37° C. Cells are induced with PPT-B (1 μM) for 7 hours to detectactivity. After incubation the cells are washed twice inphosphate-buffered saline, lysed in assay buffer (270 μM coenzyme A, 530μM ATP in 20 mM tris-HCl, pH 7.8, 1.07 mM (MgCO₃)₄ Mg(OH)₂ 5H₂ O, 2.67mM MgSO₄, 0.1 mM EDTA disodium dihydrate, and 33 mM dithiothreitol)containing 5 μM luciferin. Luciferase activity is measured in aluminometer (ML-1000, Dynatech Laboratories) and is linear between 10 nMand 0.1 mM NKB (Stratowa, C. et al. (1995) supra).

XI Production of PPT-B Specific Antibodies

PPT-B that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO:2 is analyzed using DNASTARsoftware (DNASTAR Inc.) to determine regions of high immunogenicity anda corresponding oligopeptide is synthesized and used to raise antibodiesby means known to those of skill in the art. Selection of appropriateepitopes, such as those near the C-terminus or in hydrophilic regions,is described by Ausubel et al. (supra), and others.

Typically, the oligopeptides are 15 residues in length, synthesizedusing an Applied Biosystems 431A peptide synthesizer usingfmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma,St. Louis, Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimideester (MBS; Ausubel et al., supra). Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. The resultingantisera are tested for antipeptide activity, for example, by bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radio iodinated, goat anti-rabbitIgG.

XII Purification of Naturally Occurring PPT-B Using Specific Antibodies

Naturally occurring or recombinant PPT-B is substantially purified byimmunoaffinity chromatography using antibodies specific for PPT-B. Animmunoaffinity column is constructed by covalently coupling PPT-Bantibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

Media containing PPT-B is passed over the immunoaffinity column, and thecolumn is washed under conditions that allow the preferential absorbanceof PPT-B (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/PPT-B binding (eg, a buffer of pH 2-3 or a high concentrationof a chaotrope, such as urea or thiocyanate ion), and PPT-B iscollected.

XIII Identification of Molecules Which Interact with PPT-B

PPT-B or biologically active fragments thereof are labeled with ¹²⁵ IBolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133:529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled PPT-B, washed and any wells withlabeled PPT-B complex are assayed. Data obtained using differentconcentrations of PPT-B are used to calculate values for the number,affinity, and association of PPT-B with the candidate molecules.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled in*molecular biology or related fields are intended to be within the scopeof the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 4                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 122 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -    (vii) IMMEDIATE SOURCE:                                                            (A) LIBRARY: BRAITUT03                                                        (B) CLONE: 2109906                                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - Met Arg Ile Met Leu Leu Phe Thr Ala Ile Le - #u Ala Phe Ser Leu Ala         #                15                                                           - Gln Ser Phe Gly Ala Val Cys Lys Glu Pro Gl - #n Glu Glu Val Val Pro         #            30                                                               - Gly Gly Gly Arg Ser Lys Arg Asp Pro Asp Le - #u Tyr Gln Leu Leu Gln         #        45                                                                   - Arg Leu Phe Lys Ser His Ser Ser Leu Glu Gl - #y Leu Leu Lys Ala Leu         #    60                                                                       - Ser Gln Ala Ser Thr Asp Pro Lys Glu Ser Th - #r Ser Pro Glu Lys Arg         #80                                                                           - Asp Met His Asp Phe Phe Val Gly Leu Met Gl - #y Lys Arg Ser Val Gln         #                95                                                           - Pro Asp Ser Pro Thr Glu Met Xaa Asn Gln Gl - #u Asn Val Pro Ser Phe         #           110                                                               - Gly Ile Leu Lys Tyr Pro Pro Arg Ala Glu                                     #       120                                                                   - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 754 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -    (vii) IMMEDIATE SOURCE:                                                            (A) LIBRARY: BRAITUT03                                                        (B) CLONE: 2109906                                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - AGAGCCTTTA TCAGGGAGCT GGGACTGAGT GACTGCAGCC TTCCTAGATC CC - #CTCCACTC        60                                                                           - GGTTTCTCTC TTTGCAGGAG CACCGGCAGC ACCAGTGTGT GAGGGGAGCA GG - #CAGCGGTC       120                                                                           - CTAGCCAGTT CCTTGATCCT GCCAGACCAC CCAGCCCCCG GCACAGAGCT GC - #TCCACAGG       180                                                                           - CACCATGAGG ATCATGCTGC TATTCACAGC CATCCTGGCC TTCAGCCTAG CT - #CAGAGCTT       240                                                                           - TGGGGCTGTC TGTAAGGAGC CACAGGAGGA GGTGGTTCCT GGCGGGGGCC GC - #AGCAAGAG       300                                                                           - GGATCCAGAT CTCTACCAGC TGCTCCAGAG ACTCTTCAAA AGCCACTCAT CT - #CTGGAGGG       360                                                                           - ATTGCTCAAA GCCCTGAGCC AGGCTAGCAC AGATCCTAAG GAATCAACAT CT - #CCCGAGAA       420                                                                           - ACGTGACATG CATGACTTCT TTGTGGGACT TATGGGCAAG AGGAGCGTCC AG - #CCAGACTC       480                                                                           - TCCTACTGAG ATGTNGAATC AAGAGAACGT CCCCAGCTTT GGCATCCTCA AG - #TATCCCCC       540                                                                           - GAGAGCAGAA TAGGTACTCC ACTTCCGGAC TCCTGGACTG CATTAGGAAG AC - #CTCNTTCC       600                                                                           - CTGTCCCAAT CCCCAGGTGC GCACGCTCCT GTTACCCTTT CTCTTCCCTG TT - #CTTTGTAA       660                                                                           - CATTCTTGTG CTTTGACTCC TTCTCCATCT TTTNCTACCT NGACCCTGGG TG - #TGGAAACT       720                                                                           #      754         CCCA ACCCCAATGG GCAT                                       - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 126 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -    (vii) IMMEDIATE SOURCE:                                                            (A) LIBRARY: GenBank                                                          (B) CLONE: 163590                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - Met Arg Ser Thr Leu Leu Phe Ala Val Ile Le - #u Ala Leu Ser Ser Ala         #                15                                                           - Arg Ser Leu Gly Ala Val Cys Glu Glu Ser Gl - #n Glu Gln Val Val Pro         #            30                                                               - Gly Gly Gly His Ser Lys Lys Asp Ser Asn Le - #u Tyr Gln Leu Pro Pro         #        45                                                                   - Ser Leu Leu Arg Arg Leu Tyr Asp Ser Arg Va - #l Val Ser Leu Asp Gly         #    60                                                                       - Leu Leu Lys Met Leu Ser Lys Ala Ser Val Gl - #y Pro Lys Glu Ser Pro         #80                                                                           - Leu Pro Gln Lys Arg Asp Met His Asp Phe Ph - #e Val Gly Leu Met Gly         #                95                                                           - Lys Arg Asn Leu Gln Pro Asp Thr Pro Val As - #p Ile Asn Gln Glu Asn         #           110                                                               - Ile Pro Ser Phe Gly Thr Phe Lys Tyr Pro Pr - #o Ser Val Glu                 #       125                                                                   - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 116 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -    (vii) IMMEDIATE SOURCE:                                                            (A) LIBRARY: GenBank                                                          (B) CLONE: 205725                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Met Arg Ser Ala Met Leu Phe Ala Ala Val Le - #u Ala Leu Ser Leu Ala         #                15                                                           - Trp Thr Phe Gly Ala Ala Cys Glu Glu Pro Gl - #n Glu Gln Gly Gly Arg         #            30                                                               - Leu Ser Lys Asp Ser Asp Leu Ser Leu Leu Pr - #o Pro Pro Leu Leu Arg         #        45                                                                   - Arg Leu Tyr Asp Ser Arg Ser Ile Ser Leu Gl - #u Gly Leu Leu Lys Val         #    60                                                                       - Leu Ser Lys Ala Ser Val Gly Pro Lys Glu Th - #r Ser Leu Pro Gln Lys         #80                                                                           - Arg Asp Met His Asp Phe Phe Val Gly Leu Me - #t Gly Lys Arg Asn Ser         #                95                                                           - Gln Pro Asp Thr Pro Ala Asp Val Val Glu Gl - #u Asn Thr Pro Ser Phe         #           110                                                               - Gly Val Leu Lys                                                                     115                                                                   __________________________________________________________________________

What is claimed is:
 1. A substantially purified polypeptide comprisingthe amino acid sequence selected from the group consisting of SEQ IDNO:1, a fragment of SEQ ID NO:1 from residue 1 through residue 79, and afragment of SEQ ID NO:1 from residue 94 through residue
 122. 2. Acomposition comprising the polypeptide of claim 1 in conjunction with asuitable pharmaceutical carrier.